This project proposes a series of in vivo experiments to test and improve the muscle-specific expression of viral vector-delivered micro-, mini-, and full-length dystrophin, as well as a variety of compensatory proteins which will be virally delivered to the dystrophic muscles of mdx4cv mice. Regulatory cassettes of different sizes and expression capabilities have been designed so as to be optimal for packaging into AAV, Lentiviral, and Adenoviral vectors together with therapeutic cDNAs of different sizes. The in vivo studies described in this proposal are critical for establishing the tissue specificity of regulatory gene cassettes, for improving their transcriptional activity in slow muscle fibers, for assuring that the cassettes are not expressed by immune system antigen presenting cells, and for determining whether regulatory cassettes maintain high expression levels for prolonged periods of time. After establishing the adequacy of cassette function in the expression of micro-, mini-, and fuIl-length dystrophin, the most active and tissue-specific regulatory cassettes will be used to over-express single or multiple compensatory proteins such as utrophin, alpha7-integrin, GalNac transferase-2, alpha-dystrobrevin and other members of the dystrophin-glycoprotein complex, as well as to test the beneficial function of dysferlin and other candidate proteins whose compensatory properties have yet to be examined. The overall hypothesis of these studies is that while no single therapeutic protein- especially if vector constraints require its delivery in a micro-form -- may cure DMD, the combinatorial over-expression of proteins that partially substitute for or circumvent aspects of dystrophin's function, and that repair consequences of it absence, may well ameliorate the progression of muscle degeneration, and may change the course of DMD such that the disease phenotype resembles mild forms of Becker muscular dystrophy. This would represent a major improvement for persons with severe DMD.